CA1329561C - Potyvirus coat protein genes and plants transformed therewith - Google Patents

Abstract

ABSTRACT
The present invention relates to the coat protein genes of Papaya Ringspot Virus Strain papaya ringspot (PRV-p), Watermelon Mosaic Virus II (WMVII), and Zucchini Yellow Mosaic Virus (ZYMV); to expression vectors which contain a coat protein gene for PVP-p, WMVII
or ZYMV, and, additionally, the necessary genetic regulatory sequences needed for expression of a gene transferred into a plant;
to bacterial or plant cells which are transformed with an expression vector containing the PVP-p, WMVII or ZYMV coat protein genes; to transgenic plants which are produced from plant cells transformed with an expression vector containing the coat protein gene from PVP-p, WMVII or ZYMV; and to a process of producing transgenic plants which have increased resistance to viral infection.

Description

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4658.P CNl POTYVIRUS COAT PROTEIN GENES AND PLANTS TRANSFORMED THEREWITH
FIELD OF INVENTION
The present invention relates to the coat protein genes of potyviruses. More specifically the invention relates to a process for preparing a coat protein ~ene from a potyvirus as well as its incorporation into a transfer vector, and its use in producing transformed plant cells and transformed plants which are resistant to viral infections by the particular potyvirus and relate~ viruses from which the gene ~s derived.
BACKGROUND OF THE INVENTIO~
Potyviruses are a distinct group of plant viruses which are pathogenic to various crops. Potyviruses include watermelon mosaic virus II (UMVII); papaya ringspot virus strains papaya ringspot and watermelon mosaic I (PRV-p and PRV-w), two closely related members of the plant potyvirus group which were at one time classified as distinct virus types, but .~re p~esently classified as different strains of the same virus; zucchini yellow mosaic virus (ZYMV); And many others. These viruses consist of flexous, filamentous particles of dimensions approximately 780 X 12 nanometers. The viral particles contain a single-stranded RNA ~genome containing about 10,000 nucleotides of positive (+, coding, or sense) polarlty. Translation of the RNA genome of potyviruses shows that the RNA encodes a single large polyprotein of about 330 kD. This polyprotein contains several proteins, one of which is a 49kD protease that is specific for the cleavage of the polyprotei~ into at least six (6) other psptides.
One of the proteins contained within this polyprotein is a 35kD
capsid or coat protein which coats and protects the viral RNA from degradation.
The genome organization of several viruses belonging to the potyvirus family group has been studied in detail, in particular eobacco etch virus, tobacco vein mottling virus and pepper mottle virus. I~ each case, the location of the coat protein gene has been at the 3'-end of the RNA, just prior to a stretch of (200 to 300 bases) ter~inal adenine nucleotides residues. The location of the 49 kD protease gene appears to be conserved in these viruses. In the tobacco etch virus, the protease cleavage site has been determined to be the dipeptide Gln-Ser, Gln-Gly or Gln-Ala. Conservation of these dipeptides as the cleavage sites in these viral polyproteins is - :
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apparent from the sequences of the above-listed potyviruses.
Expression of the coat protein genes from tobacco ~osaic virus, alfalfa mosaic virus, cucu~ber mosaic virus, and potato virus X in transgenic plants has resulted in plants which are resistant to infection by the respective ~irus. In order to produce such transgenic plants, the coat protein gene must be inserted into the genome of ~he plant. Furthermore, the coat protein gene must contain all the genetic control sequences necessary for t:he expression of the gene after it has been inco~porated into the plant genome.
sincs the coat protein of a potyvirus is produced by the post translational processing of a polyprotein, the coat protein gene isolated from viral RMA does not contain the genetic regulatory sequences needed for gene expression. The coat protein gene does not contaln the t~an3c~ipt~0n and translation signals nscsssary for its expression once transferred and integrated into a plant g~nome. It must, thereiore, be engineered to contain a plant expressible promoter, a translation initiation codon ~ATG) and a plant functional poly(A) addition signal (AATAAA) 3' of its translation termination codon.
In the present invention, the nucleotide sequences of the coat protein gen~s for WMV-II, PRV-p and ZYMV have be~n deter~ined, and the genes have been inserted into expression vectors to supply them with the necessary genetic regulatory sequences so that the genes can be expressed ~hen incorporsted into a plant genome. Plant cells are transformed ~ith the vactor const~uct and the pl~nt cells are inducsd to regenerat~. The resulting plants contain the coat protein genes ant produce the coat protein. The production of the protein confers upon the plant an increased resistance to infection by the virus from which the coat protein gene was derived.
INFORMATION DISCLOSURE
Europesn patent application EP 0 223 452 describes plants that ar~ resistant to v~ral disease~ and methods for producing them. The process described co~prises the steps of transforming a plant with a DNA insert comprising a pro~oter, a DNA ~equence deri~ed from ehe ~irus, and a poly~A) addition sequence.
PCT patent application PCT/US86/00514 refers generally to a method of conferring resistance to a parasite to a host of the parasite.

'-, , ~ , ,' Allison et al. (1985) "Biochemical Analysis of the Capsid Protein Gene and Capsid Protein of Tobacco Etch Virus: ~-Terminal Amino Acids Are Located on the Virion's Surface", Virology 147:309-316, describe the nucleotide sequence at the 3' end of the tobacco etch virus genome encoding the capsid protein. Homology to the sequence encoding the capsid protein of Pepper mottle virus is reported.
Allison et al. (1986) "The Nucleotide Sequence .of the Coding Region of Tobacco Etch Virus Genomic RNA: Evidence for the Synthesis of a Single Polyprotein", Virology 154:9-20 describe the genome organization of the tobacco etch virus.
Carrington, J.C. and Dougherty, ~.G. (1987) "Small nuclear inclusion protein encoded by a plant potyvirus genome is a protease", `~
J. Virology 61:2540-2548, disclose that the viral RNA of tobacco etch virus encodes the 49K p~otease responsible for cleavage of the polyprotsin produced when the viral RNA is translated.
Dodds ot al. (1985) "Cross Protection between strains of cucu~ber mosaic virus: effect of ho3t and type of inoculum on accumulatlon of virions and double-stranded RNA of the challenge strain", Virology 144:301-309, describe increased resistance to challenge by virus conferred to a plant by infection of a different strain of vi~us.
Dougherty, W.G. et al. (1985) "Nucleotide Sequence at the 3' Terminus of Pepper M~ttle Virus Genomic RNA: Evidence for an Alterna-tive Mode of Potyvirus Capsid Protein Gene Organization", Virology 146:282-291, report the nucleotide sequence of the 3' terminus of the viral RNA genome of pepper mottle virus.
Dougherty, W.G. et al. (198fl) "Biochemical and mutational analy~is of plant virus polyprotein cleavage site", EMBO J.
7:1281-1287, describe the conservation of the proteolytic cleavage site among geographically distinct lsolates of tobacco etch virus.
Dougherty, ~. G. and Carrington, J. C. (1988) "Expression and function of potyviral gene products", Ann. Rev. Phytopathol.
26:123-143, describe po~yviruses and some of the similarities the members of the group have with each another.
~ ggenberger, A. L. e~ al. (1989j "The nucleotide ~equence of a Soybean Mosaic Virus Coat Protein rsgion and i~s expression in Escherichia coli, Agrobacterium tumefaciens, and tobacco callus", .: ' ' , :
-; ~ ~

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-4- 1329~1 Virology, in press, disclose the nucleotide sequence of the coat protein gene for soy bean mosaic virus.
Hinchee, M. A. W. et al (1988) "Production of transgenic soybean plants using Agrobacterium-mediatQd DNA transfer", Bio~tech.
6:915 921, disclose the production of transgenic soybean plants which were transformed with A. tumerfacien plasmid~ that conferred either Kanamycin resistance/~-glucuronidase activlty or Kana~ycin resistance/glyphosphate tolerance.
Kozak, M. (1986) "Point mutations define a sequence flanking ths AUG initiator codon that modulates translation by eukaryotic ribo-somes", Cell 44:283-292, discloses the optimal sequence around the ATG initiator codon of the preproinsulin gene for initiation by eukaryotic ribosomes.
Loesch-Fries et al. (1987) "Expression of alfalfa mosaic virus RNA 4 in transgenic plants confers ~irus resistance", EMB0 J
6:1845-1851, disclose that expres~ion of the coat protein gene of alfalfa mosaic virus in transgenic pl2nts confars resistance to infection by the virus.
Pietrzak et al. (1986) "Expression in plants of two bacterial Z0 antibiotic resistant genes after protoplast transformation with a new plant axpression vectorn, Nucleic Acids R~search 14:5857-5868, dis-close expression in plants of foreign genes introduced into the plant using an expression vector containing a ~ovable expression cassette consisting of the Cauliflower mosaic virus 35S promoter and tranecription ter~lnator seperated by a polyllnker containing several unique restriction SitQ8.
Powell-Abel et al. (1986) "Delay of disease de~elopment in transgenic plants that express the tobacco mosaic virus cost protein gene", Science 232:738-743, disclose increased resistance toward infection by tobacco mosaic virus in transgenic plants containing the coat protein gene ~ro~ tobaoco mosaic virus.
Quemada, H. D. et al. (1989) "The nucleotide sequences oE cDNA
clones from RNA3 of Cucumber Mosaic Virus strains C and WL", J. Gen.
Virol. 70:1065-1073, reports the nucleotide sequences of cDNA clones fro~ RNA3 of Cucumber Mosaic Virus strains C and WL and co~pares them to each other and other strains for homology.
Shukla, D. D. et al. (1986) "Coat Protelns of Potyviruses", Virology 152:118-125, discloses the amino acid sequence of the po~ato .. . .
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-5~ 13~9~61 virus Y ooat protein.
Shukla, D. D. et al. (1988) "The N and C termini of the Coat Proteins of Potyvir~ses Are Surface-located and the N Terminus Contains the Ma~or Virus-specific Epitopes", J. Gen. Virol.
69:1497-1508, disclose th~t the N- and C-~e~ini regions of some potyvirus coat proteins are located at the surface of the viral particles. The viral particles were treated with trypsin and it was observed that tha enzyme treatment removed 30-67 amino .acids from the N-terminal and 18-20 amino acids from the C-terminal; the variations were dependent on the virus. The remaining portion of the coat protain, the c~re, ~as highly conserved among the various viruses.
Tumer et al. (1987) "Expression of alfalfa mosaic viruc coat protein g~ne confers cross-protection in transgenic tobacco and tomato plants", EMBO J. 6:1181-1188, d$sclose transgenic tobacco and tomato pl~nts transformed with the coat protein gene of alfalfa mosaic virus display increased resistance to infection by alfalfa mosaic'virus.
Yeh and Gonsalves (1985) ~Translation of Papaya Ringspot Virus RNh in vitro: Detection`of a Possible Polyprotein That i9 Processed for Capsid Protein, Cylindrical-Inclusion Protein, and Amor-phous-Inclusion Protein", Virology 143:260-271, describe the poss~bility that the RNA genome encodes a single proprotein which undergoes post-translational processing to form the potyvirus protein product~.
The following scientific publications are of interest but not relevant:
An et al. tl985) "New cloning vehicle~ for transformation of higher plants", EMBO J. 4:277-285 describe the construction of an expreqsion plasmid which may be stably replicated in both E. coli and A. tum~rfacian~.
An, G. (1986) "Development of plant promoter expression vectors and their use fnr analysis of differential activity of nopaline synth~se pro~o~er in transformed tobacco cells", Plant Physiol.
81:86-91, repores differences in promoter activities of transferred genes ~ithin the same cells as well as in independently derived cell lines.
Bevan et al. (1983) "Structure and transcription of the nopaline synthase gene region of T-DNA", Nucleic Acids Research 11:369-385, .

.

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-6- 1329~1 disclose the DNA sequence and plant-tumor transcription pattern of a portion of DNA from A. tumerfaciens strain T37.
Depicker et al. (1982) "Nopaline synthase: transcript m~pping ~nd DNA sequence", J. Mol. Appl. Genet. 1:561-573, discloge the DNA
sequences 5' and 3' to the nos gene found ln A. tu~erfaciens.
Hepburn, A. et al. (1985~ "The use of pNJ5ooo as an intermediate vector for genetic ~anipulation of Agrobacter:Lum Ti-plà~mids", J.
General Microbio. 131:2961-2969, describe vectors which are u~ed to transfer narrow host range vectors from E. coli to A. tumerfaciens.
Klein et al., (1987) "High-~elocity microprojectiles for deli~ering mucleic acids into li~ing cells~, ~aturs 327:70-73, disclose that nucleic acids may be delivered lnto living cells usin~
acc~lerated, small tungsten balls which pierce the cells wiehout killing them.
Rlein et al., (1988) "Factors in1uencing gene delivery into Zea mays cells by high-velocity micropro~ectiles", Bio/tech. 6:559-563, disclose that two days after bombarding plant cells with a plasmid coated micropro~ectile, expression of an gena encoding an en~y~e could be detected.
Mazur, B. J. and Chui, C.-F. (1985) "Sequence of a genomic DNA
clonQ for the small subunit of ribulose bis-phosphate carboxylase-oxygenase from tobacco", Nucleic Acids Research 13:2373-2386, disclose the D~A sequence of the small subunit of ribulose bis-phosphate carboxylase-oxy~enase from tobacco.
McCabe, D. E., et al., (1988) NStable transformation of soybean (Glycine max) by particle acceleration", Bio/tech. 6:923-926, disclose expression in soybean shoots of foreign genes intro~uced into im~ature soybean seeds using DNA coated micropro~ectiles.
Olson, M. K. et al (1989) "Enhancement of heterologous polypeptids expression by alterations in the ribosome-binding-site sequencen, J. Biotech. 9:179-190, discloses the increase in gene expression of heterologous genes ln E. coli due to the presence of an AT-rich 5' untranslated r~gion.
Slightom et al. (1983~ "Complete nucleotide sequence of a French bean storage protein gene: Phaseolinn, Proc. Natl. Acad. Sci. U.S.A.
80:1897-l901, disclose the complets nucleotide sequences of the gene and the mRNA coding for a specific phaselin type French bean ma~or storage protein.

-7- 132~Sl Vilaine, F. and Casse-Delbart, F. (1987) "Independent induction of transformed roots by the TL and TR regions of the Ri plasmld of agropine type Agrobacterium rhizogenes", Mol. Gen. ~enet. 206:17-23, disclose the respective role of Tl- and TR-DNA in root induction by S agropinc type Agrobacteri~m rhizogenes Ri plasmicls.
None of thes~ documents, either alone or taken tog~ther, teaches or suggests the instant invention which relates to poty~lrus coat protein genes and plants transformed therewith. .-SUMMARY OF THE INVENTION
The present invention relates to the coat protein genes of Papaya Ringspot Virus Strain papaya ringspot (PRV-p), Watermelon Mossic Virus II (~MVII), and Zucchin~ Yellow Mosaic Virus (ZYMV~.
The present lnvention relates to a reco~binant DNA molecule which encodes a potyvirus coat protein. The present invention relatas to a recombinant D~A molecule comprising a potyvirus coat protein gene operably linked to genetic regulatory sequences neccssary for gene expression.
The present invention relates to expression vectors which contain a coat protein gene for potyviruses, and, additionally, the necessary genetic r~gulatory sequ~nces needed for expression of a gene transferred into a plant. The prssent invention also relatss to bacterial or plant cells which are transformed with an expression vector containing the coat protein genes. Furthermore, the present invention relates to transgenic plants which are produced from plant cells transformed with an expression vector containing the coat protein gene from potyviruses. In addition, the present invention relates to a process of producing transgenic plants which have increased resistance to viral infection.
DETAILED DESCRIPTION OF THE INVE~TION
Charts 1, 2 and 3 contain DNA nucleotide sequences of the coat protein genes of PRV-p, W~VII and ZYMV, respectively. Charts 4 and 5 compare the nucleotide sequences of various coat protAln genes.
Charts 6-14 are set forth to illustrate the con~tructions of this invention. Certain conventions are used to illustrate plasmids and DNA fragments as follows:
(1) The single line figures represent both circular and linear double-stranded DNA.
(2) Asterisks (*) indicate that the molecule represented is .
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circular. Lack of an asterisk indicates the ~olecule is linear.
(3) Junctions between nàtural boundaries of functional com-ponents are indicated by vertical lines along the horizontal lines.
(4) Genes or functional components are indicated below the horizontal lines.
(5) Restriction sites are indicated above the horizontal lines.
(6) Distances between genes and restriction sites are not to scale. The figures show the relative positions only unless indicated otherwise.
(7) The following abbreviations are used to denote function and components:
a) PCà ~ CaMV35S promoter;
b) Ic ~ CMV intergenic region, the intergenic region comprising the initiation codon and AT rich 5' untranslated region;
c) Sca - CaMV35S poly(A) addition si~nal; and d) Nos - Nos nptII gene.
Most of the recombinant DNA methods employed in pràcticLng the present invention are standard procedures, well known to those skilled in the art, and described in detail ln, for example, European Patent Application Publication Number 223452, published November 29, 1986. Enzymes are obtained from commercial so~rces and are used according to the vendor's recommendatlons or other variations known in the art.
General references containing such standard technlques include the following: R. ~u, ed. (1979) Methods in Enzymology, Vol. 68; J. H.
Miller (1972) Experiments in Molecular Genetics; T. Maniatis et al.
(1982) Molecular Cloning: A Laboratory Manual; D. M. Glove~, ed.
~1985) DNA Cloning Vol. II; H.G. Polites and K.R. Marotti (1987) "A
step-wise protocol for cDNA synthesis". Biotechniques 4-514-520;
S.B. Gelvin and R.A. Schilperoort, eds. Introduction, Expression, and Analysis of Gene Products in Plants.

For the purposes of the present disclosure the following definitions apply.
"Promoter" means a promoter which is functional in the host ., .
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plant.
"Initiation region" includes the initiation codon and nucleotides flanking the initiation codon.
"Operably linked" refers to the linking of nucleotide regions encoding specific genecic information such that the nucleotide regions are conti~uous, the functionality of the region is preserved and will perform its function relative the the other regions as part of a functional unit.
"AT rich 5' untranslated region" is a nucleotide sequence composed of at least 60% adenine or thymine nucleoeides.
"Untranslated flanking region" refers to nucleotide sequences which are 3' of the termlnation codon and end at the poly(A) addition signal. These sequences enhance production of the peptide encoded by the upstream gene.
I'Vector" is a vehicle by means of which DNA fragments can be introduced lnto host organisms.
"Expre~sion vector" is a vehicls by means of which DNA ~ragments that contain sufficient genetic information and can, therefore, be expressed by the host, can be introduced into host organisms.
"Antipathogen gene" is a gene which encodes a DMA sequsnce which is either the antisense sequence of a pathogenic gene or the antipathogenic gene encodes a peptide whose presence in an organism confers an increased resistence to a pathogen.
To practice the present invention, the coat protein gene of a ~5 virus ~ust be isolated from the viral genome and inserted into a vector containing the genetic regulatory sequences necessary to expres~ the inserted gene. Accordingly, a vec~or must be constructed to provide the regulatory sequences such that they will be functional upon inserting a desired gene. When the expression vector/insert construct is assembled, it is used to transform plant cells which are then used to ragenerate plants. These transgenic plants carry the ~iral gene in the expression vector/insert construct. The gene is sxpressed in the plant and increased resistanoe to viral infection is conferred thereby.
Several different courses exist eO isolate the coat protein gene. To do so, one having ordinary skill in eh~ art can use information about the ~enome organization of potyviruses to locate and isolate the coat protein ~ene. The coat protein gene is located ..

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at the 3' end of the RNA, just prior to a stretch of about 200-300 zdenine nucleotide residues. Additionally, the information related to protaolytic cleavage sites is used to determine the N-terminus of the potyvirus coat protein gene. The protease recognition sites are conserved in the poty~iruses and have been det,2rmined to be either the dipeptide Gln-Ser, Gln-Gly or Gln-Ala. The nucleotide sequences which encode thess dipeptides can be deter~ined.
Using methods well known in the art, a quantity oi virus is grown up ~nd harve~ted. The viral RNA i9 then seperated and the coat protein gene can be lsolated using a number of known procedures. A
cDNA library is created using the viral RNA. The methods follow~d to do thi are well known in the art. The viral RNA is treated with re~erse transcriptase and a complementary DNA molecule is produced.
A DNA complement of the complementary DNA molecule is produced and that sequence represents a DNA copy of the original viral RNA
molecule. Thu5, a double stranded DNA molecule is generated which contains the sequence information of the vlral RNA. These DNA
molecules can be cloned in E. coli plasmid vectors after the additions of restriction enzyme linker molecules by DNA ligase. The various frag~ents are inserted into cloning vectors which are then used to transfor~ E. coli and create a cDNA library.
Since the coat protein gene is located ~ust 5' to the polyA
region, oligonucleotides that can hybridize to the polyA region can be us~d as hybridization probes to screen the c~NA library and deter~ine if any of the transformed bacteria contain DNA fragments with sequences codin~ for the polyA region. The cDNA inserts in any bacterial colonias which contain this region can be sequenced. The coat protein gene is present in its entirety in colonies which have sequenees that extend 5' to the seq~en e which encodes the proteo-30 lyeic clea~age site described above.
Ale~rn~tively, cDNA fragments m~y be inserted into expression vectors. Antibodies against the coat proteln may be used to screen the cDNA expression library and the gene can be isolated from colonies which express the protein.
U~ing the seguences disclosed in Charts 1, 2 and 3, the coat protein genes ~or the respective viruses may be synthesized chemically by methods well known in the art. Alterna~ively, the information in Charts 1, 2 and 3 may be used to synthesize , " '` ' '. , :, ' ,` , ' "' ''~ - ' -` 132~1~61 oligonucleotides which can be used as probes to screen a cDNA
library.
The nucleotide sequences of the coat protein genes for WMV-II, PRV-p and ZYMV have be0n determined and the gen~es have been inserted into expression vectors. The expression vectors contain the necessary genetic regulatory sequences for expression of an inserted gene. The coat protein gene is inserted such that ~hose regula~ory sequences are functional so that the genes c~m be expressed when incorporated into a plant genome.
In order to express the viral gene, the necessary genetlc regulatory s~quences must be provided. Since the coat protein of a potyviru~ is produced by the post translational processing of a polyprotein, the cost protein gene isolated from viral RNA does not contain the genetic regulatory sequences needed ~or gene expression.
The coat protein gen~ does not contain the transcription and tran~lation signal~ necessary for its expression once transferred and integrated into ~ plant genome. It must, therefore, be engineered to contain a plant expressible promoter, a translation initiation codon (ATG) and a plant ~unctional poly(A) addition signal (AATAAA) 3' of its translation ter~ination codon. In the present lnvention, the coat protein is inserted into a vector which contains a cloning site for insertion 3' of the initiation codon and 5' of the poly(A) si~nal. The promoter is 5' of the initiation codon such that when a structural gene is inserted at the cloning site, a functional unit is for~ed in.which the inserted gene i9 expressed under ehe control of the various ~enetic regulatory sequences.
In the preferred embodiment of the present invention, additional genetic regulatory sequences are provided. As described above, an expression vector must contain a promoter, an i~tiation codon and a poly(A) addition signal. In order to get a higher level of expr2ssion, untranslat~d regions 5' and 3' to the inserted genes are provided. Furthermore, certain sequences flanking th~ in~tiation codon optimize expression. The promoter used is one that is chosen for high level expression.
A 5' untranslated region which results in higher level expres-sion of an inserted gene is provided do~nstrea~ fro~ the promoter and upstream from the initiation codon. This region contains at least 60~ of the sequence a Adenine and Thymine. There is a statistical .

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bias for expression when such an AT rich region is located between the promoter and intiation codon. This preference is utilized in the preferred embodiment of the present inven~ion by inclusion of an AT
rich 5' untranslated r0gion intermediate of the pro~oter and initi-ation codon.
The prefarred embodiment of the present invention also contains sp2cific nucleotide sequence flanking the initiation codon. This preferred sequence, termed Kozak's ele~ent, is AAXXATGG wherein X
represents any of the four nucleotides. The prasence of the initiation codon following Kozak's rule results in higher level expression when used in an expression vector. In the preferred embodi~ent of the present invention, the small subunit from the SS
RUBISC0 contains an initiation codon in which Rozak's element is used.
Furthermore, the prefarred embodimant of the present invention contain~ a 3' untranslated region downstream from the cloning site where the coat protein gene i9 inserted and upstream from the poly(A) addition signal. The sequence of thi~ 3' untranslated re~ion results in a statistical bias for protein production. The sequence promotes high level expres~ion. The poly(A) addition signal is found directly downstrea~ from the 3' untranslated reglon and can be derived from the same source. In the preferred embodiment of the present invention, the 3' untranslated region and poly(A) addition signal are derived from CaMV 35S gene or the phaseolin seed storage protein gene.
The poly~A) addition signal from CaMV, nopaline synthase, octopine synthase, bean storage protein, and SS RUBISC0 genes are also suitable for this construction. Several promoters which unction in plants are available, but the best promoters are the constitutive promvter from cauliflower mosaic virus (CaMV, a plant DNA virus) and the s~all subunit of ribulose bis-phosphate carboxylase-oxygena~e (SS RUBISC0) gene.
Using methods well known to those skilled in the art, plant cells are transfor~ed with the vector construct and the plant cells are induced to regenerate. The resulting plants contain the coat protein genes and produce the coat protein. The production of the protein confers upon the plant an increased resistance to infection by the virus from which the coat protein gene was derived.

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13S~9~61 DESCRIPTION OF THE PREFERRED EMBODIMENTS
Example 1 Isolation of WMVII RNA
Uatermelon mosaic virus II (WMV II) was propagated in zucchini squash (Cucurbita pepo L) plants and RNA wa~ isolated by the method described by Yeh and Gonsalves ~Virology 143:260, 1985).
Exa~ple 2 Isolation of PRV-p RN~
Papaya rlngspot virus strain prv (PRV-p) w~s propagated in ~elly melon, Cucumis metuliferus (~and.) Mey. Acc. 2549 plants and RNA was isolated by the method described by Yeh and Gonsalves (Virology 143:260, 1985).
Exa~ple 3 Isolation of ZYMV RNA
Zucchini yellow mosaic virus (ZYMV) was propagated in zucchini squash (Cucurbita pepo L) plants and RNA was isolated by the method described by Yeh and Gonsalves (Virology 143:260, 1985).
Example 4 Synthesis of double-stranded cDNA
The procedure used to make double stranded cDNA fro~ isolated viral RNA is the same for all viral RN~ i~olated above. The purified RNA was sub~ected to the cDNA synt~esis protocol described by Polltes and Marotti (Biotechniques 4:514, 1986) and because this RNA contains an A-rich region at its 3'-end (similar to that found for many eukaryotic mRNAs) the procedure was straight-forward. The synthesis of double stranded cDNA was also done as described by Polites and Marotti. Aft~r double-stranded cDNA was synehesized, it was purified by pass~ge through a G-lOO Sephade~* column, precipitated with 25 ethanol, and suspended in 20 ~l of lOX EcoRI methylase buffer ~100 mM
NaCl, 100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 80 ~M S-adenosyl methionine, and 100 ~g/ml bovine serum albumin). An additional amount of S-adenosyl methionine (1 ~1 of a 32 ~M solution) was added to the reaction mixture, followed by the addition of 1 ~l (20 units) EcoRI methylase. The reaction was incubated at 37C for 30 minutes and stopped by incubation at 70C for 10 minutes. Then l ~l (5 units) of E. coli DNA polymerase I Klenow fragmen~ was added and incuba~ed at 37CC for lO minutes, followed by phenol/chlorofvrm extraction and eehanol precipitation. The pellet was washed in 70~
ethanol, then 70~ e~hanol/0.3 M sodium acetate. The pellet was dried and resuspended in 8 ~l of 0.5 ~g/~l phosphorylated EcoRI linkers (Collaborative Research, Inc., 128 Spring St., Lexington, MA 02173).
One ~1 of lOX ligase buffer (800 mM Tris-HCl ph 8.0, 200 mM MgC12.

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150 mM DTT, 10 mM ATP) and 1 ~1 of T4 DNA ligase (4 units) were added, and the reaction was incubated overnight at 15C. The ligation reaction was stopped by incubatlon at 65C for 10 minutes.
Si~ty ~1 of H20, 10 ~1 of lOX EcoRI salts (900 mM Tris-HCl pH 8.0, 100 mM MgC12, 100 mM NaCl), and 10 ~1 of EcoRI (10 units/~l) were added, and the reaction wa.~ incubated at 37C for 1 hour. The reaction was stopped by phenol/chloroform and chloroform extractions.
The reaction mixture was then size fractionated by passage through a Sephadex*G-100 column and the fractions containing the largest double stranded cDNA molecules were concsntrated by butanol extractions, precipitated with ethanol, and resuspended in 10 ~1 of H20. Five ~1 of the double stranded cDNAs was added to 0.5 ~g of pUCl9 DNA (which had been previously treated with phosphatase to remove the 5' phosphates), 1 ~1 of lOX ligase buffer, and 1 ~1 of T4 ligase, and the reaction was incubated at 15C for 16 hours. The resulting ligated p~Cl9-coat protein gene double stranded cDNA molecules were transformed into E.coli host cells as described by the manufacturer (Bethesda Research Laboratories, Inc., Gaitharsburg, MD 20877) and plated on medium containing 50 ~g/ml ampicillin, 0.04 mM IPTG, and 0.004~ X-Gal. Bacterial colonies showing no blue color were selected for further analysis. Clones containing the 3'-region and possibly the coat protein gene were identified by hybridization against a 3~P-labeled oligo-dT. Bacterial colonies showing hybridization to this probe should contain at least the poly(A) region of the particular potyvirus genome. Several of the hybrldizing bacterial clones were selected and plasmid DNAs were isolated accordlng to methods known to those skilled in the art.
Example 5 Identification of the PRV-p Coat Protein Gene Several of the cloned cDNAs of PVP-p RNA were sequenced by the chemical DNA sequencing method described by Maxam and Gilbert (Methods of Enzymology 65:499, 1980). Based on this information and comparative analysis wi~h other potyviruses clone number pPRV-117 ~as found to contain a complete copy of the PRV-p coat proteln gene. The N-terminus of the coat protein was identified by the location o~ the dipeptide sequence Gln-Ser. The length of the PRV-p coat protein gene coding region is consistent with a gene encoding a protein of about 33 kDal. The sequence of the PRV-p coat protein gene and ; ~, protein are shown in Chart 1. In additlon, comparison of this 'i ~
*Trade-mark ,' : . ,. : : .

~32~5~

sequence with that of the related virus PRV-w described by Nagel and Heibert (Virology 143:435, 1985~ shows that the two coat protein genes share 98~ nucleotide and amino acid similarities (Chart 4).
Because these two viruses share extensive ident:Lties in their coat proteins, expression of the coat proteln gene from PRV-p is expected to yield plants resistant to both PRV-p and PRV-w.
Example 6 Construction of a Plant-expressible P~V-p Coat Protein Gene Csssette with CaMV 35S Promoter and Polyadenylation Si~nal and CMV 5' Untranslated Region and Translation Initiator ATG.
Attachment of the necessary plant regulatory signals to the PRV-p coat protein gene was acco~plished by constructing a translational fusion with a clone originally designed for the expresslon of the CMV
coat protein gene, using clone pU~1813/CPl9. Plasmid pUC1~13/CPl9 is a vector suitable for agrobacterium medi~ted gene transfer. An EcoRI-EcoRI fragment was removed from pDH51/CPl9 and placed into the EcoRI site of the plasmid, pUC1813 (available from Robert K., Department o~ Chemistry, Washin~ton State Uhiversity, Pullman, Washington), creating plasmid pVC1813/CPl9. Plasmid pUC1813/CPl9 was described in Wo 89/5858, published June 29, 1989.
This trans- ~r lationsl fusion clone was constructed by first identifying two restriction enzyme sites within clone pUCl813/CPl9. One site (Tthlll I) is located between amino aclds 13 to 17 while the other site (BstX
I) is located near the end of the 3'-untranslated region of the CMV
coat protein gene.
Addition of these specific restriction enzyme sites to the P~V-p coat protein gene was accomplished by the polymerase chain reaction technique, using an instrument and Taq polymerase purchased from Perkin Elmer-Cetus, Emeryville, Ca. Specifically, two respective 5' and 3' oligomers (CGACGTCGTCAGTCCAAG MTGAAGCTGTC, cvntaining a Tthlll I site and (CCCACGAAAGTGGGGTGAAACAGGGTCGAGTCAG, contalning a BstX I
site), sharing at least 20 nucleotides with the PRV-p coat protein gene were uaed to prime syn~hesLs and gene amplification of the coae protein gene. After synthesis, the amplified fragments were digested with Tthlll I and BstX I to expose the sites.
As shown in Chart 6, pVC1813/CPl9 is the expression vector wh$ch contains the CMV coat protein gene. Plasmid pUC1813/CPl9 contains Tthlll I and BstX I sites.

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The di~ested, amplified fragments are ligated into the respec-tive exposed sites of pUC1813/CPl9 and the expected new construction was idantifisd using methods known to those skilled in the art.
Polymerase chain reaction technigues were used to ampliiy PRV-P coat protein gene containing the TthlllI and BstXI sites. The plasmid pUC1813/CPl9 and PR~-P coat protein gene fragments were digested with TthlllI and BstXI and ligated to each other. The resulting clone, designated pUC1813/CPl9-PRVexp, was sub~ected t~ nucleotide sequencing to ensure that the cloning and gene a~plification did not introduce any detrimental artifacts. The sequence showed no arti-facts, suggesting that this plant expression cassette should be capable of expressing a P~V-p coat protsin gene which contains an additionai 16 amino acids of CMV coat protein at its N-terminus.
Example 7 Construction of a Micro T-DNA Plasmid Containin~ the Plant-expressible PRV-p Coat Protein Gene Construction.
As depicted in Chart 7, the plant expression cassette Por the P~V-p coat protein gene ~as transferred into a suitable micro T-DNA
vector which eontains the necessary Agrobacterium T-DNA transfer signals for tr~nsfer from an A~robacterium and inte~ration into a plant g~nome, and a wide host-range origin of replication (for replication in Agrobactarium). Plasmid pUClôl3/CPl9-PRVexp was digestad with Hind III and the resultng 2.2 kb insert fragment containing t~e plant-expressible cassette was removed and ligated ~nto the Hind III si~e ~f the modified Agrob~cteriu~-derived micro-vector pGA482 (modification included the addition of the ~-glucuronidase gene). The micro T-DNA vector, pGA482, is available from G. An, Institute of Biological Chemistry, Uashington State University, Pullman, WA. The resulting plasmid was designated, pGA482/G/CPl9-PRVexp and iq shown in Chart 7. This plasmid (or derivatives th~reof) ~as transferred into virulent or avirulent strains oi Agrobscterium tumefaciens or rhizogenes, such as A208, C58, LBA4404, G58Z707, A4RS, A4RS(pRiB278b)j and otherq. Strains A208 G58, LBA4404, and A4RS are available from American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD. Bacteria A4RS(pRi~278b)is available from Dr. F. Casse-Delbart, C.N.R.A., Routede Saint Cyr. F7B000, Versailles, France. Strain C58Z707 is available from Dr. A.5.Hepburn, Dept. of Agronomy, University of Illinois, Urbana, IL.

- ~ , , ` -17- ~3~
After transfer of the engineered plasmid pGA482/G/CPl9-PRVexp into Rny of the above listed Agrobacterium strains, these Agro-bacterium strains can be used to transfer and integrate within a plant genome the plant-expressible PRV-p coat protein gene contained within its T-DNA region. This transfer can be accomplished using the standard methods for T-DNA transfers which are known to those skilled in the art, or this transfer can be accomplished using the methc,ds described in a U.S. patent no. 5,169,770.

Example 8 Construction of a Plant-expression Cassette for ~xpression of.Various Genes in Transgenic Plants.
In the preferred embodiment of the present invention, the following expression cassette was constructed to provide the necessary plant regulatory signals (which include the addition of a promoter, S' untranslated region, translation initiation codon, and polyadenylation signal) to the gene inserts in order to achieve high level expression of the inserts. The expression cassette may be used to express any genes inserted therein. Accordingly, the applicability of the expression cassette goes beyond its use in expressing coat protein genes. Rather, the expression cassette may be used to express any desired protein in transgenic plants. The expression cassette is the preferred expression system for expressing viral coat protein genes in plants.
The expression cassette of the preferred embodiment contains: a constitutive promoter; a 5' untranslated region which enhances gene expression; an initiation codon which comprise Ko~ak's element; a cloning site where the gene to be expressed may be inserted to produce a functional expression unit; and a 3' untranslated region which comprises a poly(A) addition signal and untranslated flanking regions which result in a higher level of expression.
More specifically, the expression cassette which is the preferred embodiment of the present invention consists of the cauliflower mosaic virus (CaMV) 35S transcript promoter, the 5'-untranslated region of cucumber mosaic virus tC.~V), the CMV
translation initiation codon, and the CaMV polyadenylation signal.
.35 The construction of this expresslon cassette utillzed ~he Polymerase ~ ~ I

.

. ~' .

- 13295~1 Chain Reaction (PCR) technique to obtain correct position of the plant regulatory signals and the addition of convenient restriction enzyme sites which allow for the easy addition oE a coat pro~ein gene and the excision of the completed ca~sette so it can be transferred S to other plasmids.
To accomplish the construction of this expression cassette the following oligomers were synthesized: -1. 5'-GMGCTTCCGGAAACCTCCTCGGATTCC-3', contains a. HindIII site at its 5'-end and contains 21 bases which are identical to 21 bases ln the 5'-flanking region of CaMV.
2. 5'-GCCATGGTTGACTCGACTCAATTCTACGAC-3', contains a NcoI site at its 5'-end which contains a translation initiation codon which conforms to Kozak's rule~ and ha~ 21 bas~s which are identical to 21 bases in thè antisense strand of the CMV 5'-untranslated region.
3. 5'-GCCATGGTTGCGCTGAAATCACCAGTCTC-3', contains a Ncol site at its 5'-end (which contains the ~ame translation initiation codon as oligomer 2) and has 20 bases which are identica~ to-20 bases in the 3'-untranslated region of CaMV.
4. 5'-GAAGCTTGGTACCACTGGATM TGGTT-3', contains a HindIII site at its 3'-end and has a 20 base ~atch w~th the lanking DNA reg~o~ 3' of the CaMV polyadenylation site (on the antisense 3trand).
Thesa oligomers were used to amplify sequences co~tained within the CMV expres~ion clone referred to as pUC1813/CPl9, shown in Chart 6, and referred to above. As depicted in Chart 8, the PCR technique 25 was used to ampli~y the gene regulstory regions in pUC1813/CP19.
Amplification of the S'-flanking, CMV 5'-untranslated region, and CMV
initiat~on codon (which was modified to conform to Kozak's rule AAXXATGG) r~sulted in a fragment of about 400 base pairs in length and amplification of the CaMV 3-untranslated and flanking regions resulted in a fragment of about 200 base pairs i~ length. These fragment~ were digested with NcoI and HindIII, isolated from a polyacryl~ide gel, and then ligated into HindIII digested and phosphatase treated pUC18. The resulting clone is referre~ to as pl8CaMV/CNV-exp and ls shown ln ~hart 8.
Example 9 Identification of the WMVII Coat Protein Gene The cloned ~MVII cDNA insert from clone pWMVII-41-3.2 which was produced as described above, was sequenced by using both the chemical (Maxam and Gilbert, Methods of Enzymology 65:499, 1980) and enzymatic :

'~ . ~ ;. . . ' . , :

~329~
--lg-(Sanger et al., Proc. Natl. Acad. Sci. U.S.A. 74:5463, 1977) methods.
Based on this information and comparative analysis with other potyviruses, the nucleotide sequence of clone pUMVII-41-3.2 was found to contain a complete copy of the WMVII coat protein gene. The N-terminus of the coat protein was suggested by the location of thedipeptide sequence Gln-Ser. The length of the WMVII coat protein gene coding region ~281 amino acids) is consistent with a gene ~nooding a protein of about 33 kD. The sequences of this WMVII coat protsin gene and pro~ein are show~ in Chart 2. In addition, comparison of this s~quence with that obtalned from the related virus Soybean Mosaic Virus tSMV) strain N described by Egg~nberger et al.
shows that they share overall about 88% identity and excluding the N-terminal length differences th~y share abo~t 92.5~ identity, see Chart 5. Because these two vlrus coat proteins share extensive amino acid identities, expression of the coat protein gene from WMVII is expected to yield plants r~si.qtant to WMVII infection and could yield plants resistant to SMV infection.
Example 10 Construction of a Plaot-expressible WMVII Coat Protein Gen~ Cassette with Ca~V 35S Promoter and Polyadenylation Signal and CMV Intergenic Region and Translation Initiator ATG.
As depicted in Chart 9, attachment of the necessary plant regulatory signals to the WMVII coat protein gene was accomplished by using the PC~ technique to amplify the WMVII coat protein gene using oligomers which would add the necessary sltes to its 5' and 3' sequence~. Following this amplification the resulting fragment is digested with the appropriate restriction enzy~e and clone~ into the ~coI site of the above described expression cassette containing plasmld, pl8CaMV/CMV-exp. Clones containing the WMVII coat protein gene insert need only be checked to determine correct orientation with respect with the CaMV promoter. However, to ensure that no artifacts have bsen incorporated during the PCR amplification the entire coat protein gene region is checked by nucleotide sequence analysis.
To obtain the amplified WMVII coat protein gene with NcoI
restriction enzyme sites on both ends the following two oligomers were synthesized:
1. 5'-ACCATGGTGTCTTTACM TCAGGAAAAG-3', which adds a NcoI site to the 5'-end of the WMVII coat protein gene and retains the same ATG

~32~561 translation start codon which is present in the axpression cassette, pl8CaMV/CMV-exp.
~ . 5'-ACCATGGCGACCCGAAATGCTM CTGTG-3', which adds a NcoI slte to the 3'-end of the WMVII coat protein gene t this site can be S ligated into the expression cassette, pl8CaMV/CMV-exp.
The cloning of this PCR WNVII coat protein gene, U5i~g these two oligomers, into pl8CaMV/CMV-axp yields a plant expressible ~MVII gene (referred to as pl8WMVII-exp) which, following tran~cription and translation, will generatP a WMVII coat protein which is identical to that derived from the WMVII coat protein gene nucleotide sequence, see Chart 2. However, this coat protein will difer, because of necessary genetic engineering to add the ATG initiation codon and by includ~ng the last four a~ino acids of the 54 kD nuclear inclusion protein (which ls ad~acent to the Glu-Ser protease c}eavage site);
the amino acids added are Val-Ser-Leu-Glu-N-ter WMVII. The addition of these four amino acid residues should not affect the ability of this coat protein to yisld plants which are resistant to WMVII
infection~, because the N-terminal region of potyvirus coat proteins appear not to bc well conserved for either length or amino acid identity. ~owever, if this ~s found to be a problem its replacement wo~ld involve the use of a different oligomer to obtain N-t~rminal variations of the WMVII coat protein gene. The cloned construction of th~ plant expressible WMVII coat protein gene is referred to as pl8WMVII-exp, and is shown in Chart 9.
: 25 Example 11 Conseruction of:a Micro T-DNA Plasmid Containing the Plant-expressible WMVII Coat Protein Gene Construction.
As depicted in Chart 10, the plant expression cassette for the WMVII coat protein ~ene ~as transferred i~to a suitable micro-T-D~A
vector which contains the necessary Agrobacteriwm T-DNA transfer slgnals (to mediated transfer from an Agrobacteriu~ and integration into a plant genome) and wide-host range origin of replication (for replicatlon in Agrobacterium) to form plasMid pGA482/G/CPWMVII-exp.
To construct this plasmid, plasmid pl8WMVII-exp was dlgested with Hind III (which cuts within the polycloning sites of p~C18, well outside of the expression cassette), and an 1.8 kb fragment containing the plant-expressible cassette was removed ant ligated into the Hind III site of the modified Agrobacterium-derived micro-vector pGA482 (modification included the addition of the ~-~ -21- ~329~1 glucuronidase gene). The micro T-DNA vector, pGA482, is shown in Chart 7 and available from G. An, Institute o Biological Chemistry, Washington State University, Pullman, WA. The resultlng plasmid was designated, pGA482/G/CPWMVII-exp is shown in Chart 10. This plasmid (or derlvatives thereof) was transferred into virulent or avirulent strains of Agrobacterium tumefaciens or rhizogenes, such as A208, C58, LBA4404, C58Z707, A4RS, A4RS(pRiB278b), and others. Strains A208 C58, LBA4404, and A4RS are s~ailable from Americap Type Culture Collection (ATCC~, 12301 Parklawn Drive, ~ockville, MD. Bacteria A4RS(pRiB278b)is available from Dr. F. Casse-Delbart, C.N.R.A., Routede Saint Cyr. F78000, Versailles, France. Bacteria C58Z707 is available from Dr. A.G.Hepburn, Dept. of Agronomy, University of Illinols, Urbana, IL.
After transfer of the engineered plasmid pGA482~G/CPWMVII-exp into any of the above listed Agrobacterium strains, these Agrobacterium strains can be used to transfer and integrate within a plant genome the plant-expressible WMVII coat protein gene contained within its T-DNA region. This transfer can be accomplished using the standard methods for T-DNA transfers which are known to those skilled in the art, or this transfer can be accomplished using the methods described in U.S. Patent no. 5,~69,770 entitled "Agrobacterium Mediated TransEormation of GermLnsting Plant Seeds". In addition, it has recently been shown that such Agrobacteria are capable of transferring and integrating their T-DNA
regions into the genome of soybean plants. Thus these strains could be used to transfer the plant expressible ~MVII coat protein gene into the genome of soybean to develop a soybean plant line which is resistant to infection from soybean mosaic vlrus strains.
Example 12 Microprojectile Transfer of pWMVII-exp into Plant Tissues.
Recently an alternative approach for the transfer and integration of DNA into a plant genome has been developed. This technique relies on the use of micropro;ectiles on whlch the DNA
(plasmid form~ is attached. These microprojectiles are accelerated to high velocities and their momentum is used to penetrate plant cell walls and membranes. After penetration into a plant cell the attached DNA leaches off the microproJectile and is transferred to fj,,~ , the nucleus where DNA repair enzymes integrate the "free" DNA into 3~.. 1 ' .

",~' ' : , 132~

~he plant genome. In its present form the process is entirely random, but plant tissues which have been succesc~fully transformed by the plasmid DNA (or part of it) can be identified and cultured to homogeneity by the usa of selectable marker genes (such as the bacterial neomycin phosphotransferase II gene, NPTII), or reporter genes (such as the bacterial beta-glucuronidase gene, Gus).
Succes~f~l use of particle acceleration to transfor~ plants has recently been shown for soybean and the tranqfer of pl8~MVII-exp into the genome could result in obtaining soybean plants which are resistant to infections from soybean mosaic virus strains.
Th~ uie of this process for the transfer of pl8WMVII-exp can be accomplished after the addition of either plant expressible genes NPTII or Gus genes or both. Plasmids that have the nptII and Gus genes to pl8WMVII-exp are shown in Chart 11, and referred to as pl~GWMVII-~xp and pl8NGWMVII-exp. In add~tion, the construction described in Example 11 can also be used for microprojectile transfer as it already has both tho nptII and Gus genes attached to the pWMVII-exp cassette (see Chart 10). The only difficulty which the use of pGA482GG/cp~MVII-exp may encounter during transfer by the microprojectile process is due to its large si~e, about 18kb, which may have a lower efficiency transfer and such larger plasmid gensrally yield less DNA during propagat~on.
To construct plasmid pl8GWMVii-exp, plasmid pl8WMVii-exp is digested with BamHI and ligated with a 3.0 kilobase BamHI isolated fragment containing the Gus gene. To construct plasmid pl8NGWMVii-exp, the plasmid pl8GWMVli-exp is digested with SmaI and ligated with a 2.4 ~b isolated fragment containing the Nos-nptII gene generated by dig~stion with Dral and Stul.
Example 13 Identification of the ZYMV Coat Protein Gene.
The cloned ZYMY cDNA insert fro~ clone pZY~V-15, which was cloned using the method described above, was sequenced by using both the che~ical (Maxam and Gilbert, Methods of Enzymology 65:499, 1980) and enzymatic (Sanger et al., Proc. Natl. Acad. Sci. U.S.A. 74:5463, 19773 methods. Based on this infor~ation and comparative analysis with other potyviruses ~he nucleotide sequence of clone pZYMV-15 was found to contain a compLete copy of the ZYMV coat protein gene. The N-terminus of the coat protein was suggested by the location of tha dipeptlde sequence Gln-Ser which is characteristic of cleavag~ sites .: , .
.: . ~ .

~L 3 2 ~

in potyviruses (see Dougherty et al. EMB0 J. 7:1281, 1988). The length of the ZYMV coat protein gene coding region (280 amino acids) is consistent with a gene encoding a protein of about 31.3 kD. The sequences of this ZYMV coat protein gene and protein are shown in Chart 3.
Example 14 Construction of a Plant-expressible ZYMV Coat Protein Gene Casqette with CaMV 35S Promoter and Polyadenylation Signal and CNV Intergenic Region and Translation Initiator ATG.
As depicted in Chart 12, attachment of the necessary plant regulatory sign~ls to the ZYMV coat protein gene was accomplished by using the PCR technique to amplify the ZYMV coat protein gene using oligomers which would add the necessary sites to its 5' and 3' ~equences. Following this amplificatio~ the resulting frag~ent is digested with the appropriate re3tric~ion en~yme and cloned into the NcoI site of the above expression cassette containing plasmid, pUC18CP-exp. Clones containing the ZYMV coat protein gene insert need only be checked to determine correct orientation with respect with the CaMV promoter. Houever, to ensure that no artifacts have been incorporated during the PCR amplification the entire coat protein gene region is checked by nucleotide sequence analysis.
To obtain the amplified ZYMV coat protein gene with NcoI
restriction enzyme sites Gn both ends the following two oligomers were synthesized:
1. 5'-ATCATTCCATGGGCACTCMCCAACTGTGGC-3', which adds a NcoI
sita to the 5'-end of the ZYMV coat protein gene and retains the same ATG translation start codon which is present in the expression cassette, pUC18cpexp.
2. 5'-AGCTAACCATGGCTAAAGATATCAAATAAAGCTG-3', which adds a NcoI
site to ehe 3'-end of the ZYMV coat protein gene, this site can be ligated into the expre~sion cassette, pUC18cpexp.
The cloning of this PCR ZYMV coat protein gene, using these two oligomers, into pUC18cpexp yields a plant expressible ZYMV gene (referred to as pUC18cp~YMV) which following tra~scription and -translation will ~enerate a ZYMV coat protein which is identlcal to that derived from the ZYMV coat protein gene nucleotide sequence, see Chart 3. However, this coat protein w~ll differ, because of neces^
sary genetic engineering to add the ATG initiation codon fo}lowed by Gly, which is the a~ino acid 3' ad~acent to the Ser of the polyprot-.

-24- ~32~6~
ein cleavage site ~see Chart 3). The Gly a~ino acid residue was selected for the potential N-terminal zmino acid because many potyvirus coat proteins have either an Ser, Gly, or Ala at their N-terminal. Howe~qr, if this is found to be a proble~ its replacement would involve the use of a different oligomer to obtain a different N-terminal amino acid for the ZYMV coat protein. The cloned construction ~f the plant expressible ZYMV co~t protein gene is referred to pUC18cpZYMV, and is shown in Chart 12.
Example 15 Construction of a Micro T-DNA Plasmid Containing the Plant-expressible ZYMV Coat Protein Gene Construction.
Following the teachings of Example 11 with appropriate modif~cAtions, ~he construction of a ~icro T-D~A plas~id containing a plant-expressible ZYMV coat protein was constructed. Plasmid pUC18cpZYMV ~Chart 12) was digested ~ith Hind III (which cuts within the polycloning sites of pUC18, well outside of the expression cassette), and a 1.6 kb fragment containing the plant-expres~ible cassette was removed and ligatad into the ~ind III site of the micro-vector pGA482 (Chart 7). The resulting plasmid was de~ignated, pGA482GG/cpZYNV is ~hown in Chart 13.
After transfar of the enginaered plasmid pGA482GG/cpZY~V into Agrobacteriu~ strains, the Agrobacterium strains can be used to transfer and integrate within a plant genome the plant-expressible ZXMV coat protein gene contained within i~s T-DNA region. - -Example 16 Micropro~ectile Transfer of pUC18cpZYNV into Plant ~5 Tissues.
Following the teachings of Example 12, the microprojectile transfer technique can be used to intrsduce the ZYMV coat protein gens wlth ~ppropriate genetic regulatory sequences into plant tissues .
The use o$ this process f~r the transfer of pUC18cpZYMV can be accompliqhed after the addition of either plant expressible genes ~PTII or Gus genes or both. PlaRmids that have the nptII and Gus genes to pUC18cpZY~V are shown in Chart 14 and referred to as pUCl8GcpZYMV and pUC18NGcpZY~V. In addition, the construction described in ~xample 15 can also be used for microprojectile transfer as i~ already has both the nptII and Gus genes attached to the pUC18cpZYMV cassekte (see Chart 13). The only difficulty which tha use of pGA482GG/cpZYMV may encounter during transfer by the .: , . ' ' , , ~, , ,' ,: ' , 329~1 microprojectile process is due to its large size, about 18kb, which may have a lower efficiency transfer and such larger plasmid generally yield less DNA during propagation.
To construct plasmid pUC18GCPZ~V, plasmid pUC18CPZYMV is digested with BamHI and ligated to a 3.0 BamHI isolated fragment which contains the Gus gene. To construct p:lasmid pUC18GCPZYMV, plasm~d ~UC18GCPZYMV is digested with SmaI and ligated with a 2.4 kb isolated fra~ment containing the ~os nptII gene isolatQd by digestion with DraI and StuI.

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CHARTS

GtnS--rLy~A~nGluAl~vDiAi~pAlaclrLnuA~ncluLr~L~uLr~GluL~cluA~n CAGAAAGAAAAAGAAAAACAAAAACAAAAACAaAAAGAAAAACACaGTCCTAGTGACCCA
81 - ~ ~ 4 - - ~ - - - ~ 12~
ClnL~DCluLrDClul.r~CluLrsl:lnL~CluLr~CluL~-ArpGl~Al~S~rArpCl~r :
AATCATCTCTCAACTACCACAAAAACTCGACACACAG~TACAG~T¢TCAATCTTCCCACC
ArnArpV-lSorThrS-rThrL~ThrGl~rCluAr~lA~pArgA~pV~lA~nV~lClrThr ACTGC~ACTTTCACTGTTCCC~CAATTAAATCATTTACTC~TAACATCGTTCTACCCACA
S~rCl~ThrPhoT~rV-lProAr~IloL~4SorPh-ThrA~j~y~-tV-lL-uProAr~
ATTAACCCCAACACTCTCCTTA~TTTAAATCATCTTCT7CACTAC~ATCCCCAACAAATT
IloL~GlyLy~ThrVnlL~u~YnL~uA~nHi~LouLsuGlnT~rAqnProClnGlnIlo GACATTTCTAACACTCCTGCCACTCATTCACAATTTGACAACTGCTATGACCCACTGAGG
301 ~ ~ 3~0 ~pII-S-rAsnThr~r~AlrThrHi~S$rCln~h~CluLyrTrpTyrGluClyv~lAr~

31!Sl------------------4 -- -------------- --~---------- ------ --f--_ _ __ _ ___~_ _ ____ ___ ~ __ _ ____ _ _~ '120 .~nA~pT,~rClrL~uA~nA~pA~InGlulA~tGlnVnlllo~L~uA~nCl~L~u~ntV-l'rrp TCTATCGACAATCCTACATCTCCACACATATCTGCTCTCTGGOTTATCATCCATCCGCAA
C~IloCluA~nGl~hrS~rProA~pIl~S~rCl~VolTrpV~lV~t~tA~pGl~Glu ACCc~AGTTcATTATccAATcAAcccTTTGATTGAGcATccTAcTccGTcATTT~GccAA
ThrClnV~lA~pT~rPro~l~L~rProL~uIisCluHl~Ai~ThrProSorPh-Ar~Cln ATTAT~CCTCACTTTAGTAACCCCI:CACAAGCATACATTCCCAAOACAAATCCTACTGAe I1~14~t,AI-Hl~Ph~S~rADnAl~All~Clu~ trrII-Al~L~ r~A~nf~lqThr~lu -27- ~ 3 2 CHART 1 (ConCinued) AccTAcA,Tcccl:cccTATccAATcAAcAcAAArTTcAcTcAcATTAcccTcGcT~lcATAc SOl ArgTrrU~ProAr~TrrClyIl~ ArgA~nLo~ThrA-pII-5~rL~uAI-Ar~T~r CCTTTCCACTTCTATGAGGTGAATTCCAAAACACCTCATAGGCCTCCCGAAGCTCACATG
AI~PhoAYpPh~T~rGluVI~lA~nSorLy~lThrProA~?Arl~Al~Ar6~CluAl~Hi~
CACATCAACGCTGCACCGCTCCGAAACACCACTCCCAAAATGTTTCCTATGCACCGCAGT
721 --------~---------~---------~---------~---_____-~-________~ ~o CtnUotLysAlaAluAl~L~uAr~Asn'rhrS-rArgL~bhtPh-Ol~lAot.A~pClySe~r GTTAGTAACAAGCA~C~AAACACGCACACACACACAGTGGAAG~TCTCAATACACACATC
V~lS-rA~nL~GluCluA~nThrCluAr~H;~ThrVrlCluA~pV~lA~nAr~A~p~
CACTCTCTCCTGGGTATGCGCAACTAAAtACCTCCCCTTGtCTGtTTGTTCACTCTCACT
Hi~S-rL-uLouGl~ AngA~nEnd CCACCCTCTTTCACCTTATGCTACTATATAAGCATTAGAATACACAGTGCCTCCCCCACt GCTTCTATTTTACACTCACCGTACCCCTCCCTCCTTTTAGT~TTATTCGAGTTCTCTGAG

TCTccATACAcTGTGccTGccccActTCA7ATTCGACCCTCTTACAATGAGAAAAAAAAA
~ 1380 AAAAAAAAAAAAMAAAAAAAAAAAAAAAAAAAAAACCAATTCC

' :

.' . ~ '`~'' " ' ''.' ' ` ` ':

- :

-28- 1329~1 GTGTCTTT~CA~TCAGG~AAAG~AACACTTGA~AATT~GG~CGC~GGGAAAGAATC~AAG
1 6~
ValS~rLouGlns~rclyL~ysGluThrvalGlu/~snLouAspAlaGl~L~sGlusa~L~s AAAGATGccAGTGAcAAAGGGAATAAcccGcAGAAcTcGcAAGTTGGTcAGGGTAG~AG

LysAspAl~SorAspLysCl~AsnLysProGlnAsnS~rGlnV31Gl~GlnGlySerLys GAAccAAcAAAAAccGGcAcAcTc~GcAAGGATGT~AATGTTGG~TcGJ~AAGGAAAAGAA

CluProThrLysThrGlyThrV~lS~rLysAspValAsnV~lGlyS~rLysGlyLysGlu GTCCCACGACT~C~AAAGAT M CAA~GAAAATGAATCTTCCA~CAGT~GGTGGGAAAATC
181 - -- ~ --- ~ ~ -----~--------- 24 Val~roAr~LouGlnLysIloThrL~sLysUotA~nLouProThrValGlyGlyLysIlo ATtCTTAGCTT~G~CC~TTTGCTTGAGTACAAACCTAGTCAAGTTGATTTGTTTAACACT
241 ~ o Il~LouSorL~uA~pHi~LouL~uGluTyrLy~ProSorClnV~lA~pLouPh~AsnThr .
CG/~GCAACAAAiACACAATTTGAATCATGGTACAGCGCAGTCAAAGTTGACTATGATCTT
301 ---------~--- -----~---------~ -------- t - - - - - - - - ~ - - - - - - - - - ~ 360 ArgAl~ThrLysThrGlnPh~GluS~rTrpTyrS~r~l~ValLy~ValGluTyrAspL~u AATCATt;AGCAAATGGGTGTGATrATCAATGCTrTTATGGTTTGGTGTATCGATAACGGT

AsnAspGluGln~etGl~V~ M~tA~nGl~Ph~tValTrpCysIloAspAsnGly ACATC7CCACATGTCAATGGAGTGTCGGT~ATC~TCGATCGGGAAGACCAAGTTGAGTAC
~21 ~ 480 ThrSorProA.:pYal~nGl~VDlTrpV~tlMotMot,Asp~;lyCluGluGlnV;IlGluTyr CC~CTAAAGCCAATTGTTGAAAATGCAAACCCA~CTTT~AGACAAATCATGCACCATTTC
481 - - o ----- _~---_----_~__-_____ O_--___--_~----_----~ 540 ProL~uL~ProIl~V~IGluAsnAl~L~sProThrL~uArgGlnIl~tHisHisPho TC~CATGCAGCGCAAGC~TATATTGAAATGACAAACTCTGAAAGTCCCTATATGCCTAGA
541 --- ~ --~- -----~----- --o-------- ~ - ~ 60~ .
S~rA~pAlDAl~CluAlaT~rIl-GluM~tArgA~nSorGluS~rProT~rM~tProArg TACGATTACTGAGAAATTTG~GAGACAGGGAATTAGCACGCTATGCTTTTCACTTCTAT
T~rGlyLauletuArgA~nL~u~rsA~pArgGluL~tuAl~t~rgTy~Al~PhoA~pPhoTyr G~GGTTACTTCTAAAACCeCAAAr~GCGS:AAGAGAACCAAT~CCTCAAATG~AGGCCGCG
~61 -~ --~---------t-- --- ~ 720 GluV-lThrS~rLy~ThrProA~nArgAlaAraGluAl~IloAl~GlnhlotLy~Al~AI~
CCTCTCCCGCCAGTTAACAGCACCTTATTTCCACTTGATCCTAATATCTCGACCAATTCC
AI~Lou~ ClyVI~lA~nS~rA~ L~uPhoClrL-uA~pClyA$~ tS~rThrA3nSor , -29- ~329~6~
CHART 2 (Gontinued~

GAAAATACTGGG~GCCACACTGCAACGGACGTAAATCAGAATATGCATACTTTGTTGGGT
781 8s0 GluAsnThrGl~ArgHisThrAlaArgAspValAsnClnA~n~tHisThrL~uLouGI~
ATGGGTCCACCGCAGTAAAGACTAGGTAAACTCCTCACAGTTAGCATTTCGGGTCGTTAT
M~tClyProProGI nEnd A~TTTTCT~TAAT~TAACATI`.TcCcAcTTTATTTTAGTATAt:TGTr~TTT~T~ TT
901 . - ~ ~ --- ~---------~ --------~-- ~ 960 TGT~CTGTTTATGTTAGCCTCGTTTAACCACCTTTGTCTGTGCTTTAT~TTATAGTTTAT

CCGTACCAGGCAGAACCATTACAATGCCCCAGTTCTTTGTAGTCTCATTTCATCACCCTT

AATACCCGAGCTACGGTAATCTTTCTTGCCTAAA M AAAAAAAAAAAAAAAAAAA

.

-30 13295~1 GH~RT 3 ATGcTccAATc~GccAcTcAAccAAcTcTcccAGAcGcTAGAcTTAcAAAG~AAGAT~AA
MotLauGlnS~-ClrThrClnProThrV31AI-A~vAl~Ar~V~lThrLr~L~A1pLrs GAAGATGAC~AA~GGGA~A~CAAOGATTTCACACCCTCCGCCTCAGCTGAGAAAACACTA
G;luAJpA~pL,~ClrCluAtnL~ A~pPh~7'hrGl~S~rGl~SoralyClul,~oThrV-I
CTAGCTGCCAACAAAGAC~GGATGTC~ATGCTGCTTCTCATCGGAAAATTGTGCCCCCT
ValAl~AluLr~Ly~A~pL~3A~pValA~r.~l~ClrS~rHiJClyLy~IhbV~lProAr~
CTTTCOAAGATCACAAAGAAAATCTCATTCCCACGCGTC~AACCGAATCTOATACTCGAT
L~uS-rL~I IoThrLy~Lr~ S~rL~uProArgV~ IL~CI3~ArnV~ LouA~p ATCGATCATT~CCTCCAATATAACCCCCATCAAATTCAGTTAT~CAACACACGAGCGTCT
241 -----------------__--____~___~_________~_________~________ ~ 300 II~A~pH;~L-uLouCluTyrL~rProA-pGlnII-GluL-uTrrAsnThrAroAl~S-r CATCACCAATTTGCCTCTTGCTTCAACCAAGTTAACACAC~ATATGATCTOAATGATCAA
Hi~ClnClnPh~AI-S-rTrpPh~A~nClnVslLrqThrGluTrrA~pL0uA~nA~pGln CAGATCCCACTTCTCATCAACGGTTTCATGGTtTGCTCTATtCAAAATCCCACCTCACCT
3~1 ---------~----~----o---------o---------~ ------- ~---------o ~20 Gln~ ~GlyVrlVul~ 3nClrPh~tV-lTrpC~.ItoCluA8nCI~ThrSorP
GACATTAATCGACTCTCCTTTATCATG~CCG~AATGAACAAGTTCACTATCCTTTOAAA
A~pII-A~nClrVI~lTrpPho ~ ~ A~s~Gl~A~nCluGlnY~ JT,~rrProL-uL~Y
CCGATACTTCAAAAtCCAAACCCAACGCTCCCCCAAA~AATCCATCATT m CACATCCA
4al ---------~---------~_________~_________~__ ______~_________~
Pr~ V-l¢luA~nAlaL,r~roThrL~uArgCln~ tHi~Hi~PhoS-rAspAla GCGGAGCC~7ATATA6~GATc~cAAATCcAGAcccAccATAcATGCCGAcGTATacTTTG
6~ 1 A I aG I I!A I ~Ty r I loGI u~A -~A~nA I ~G I uA I a~roT~ r~Pro~roT~ ra I ~ u ,, ~

, . .

-31- ~329~61 CH~RT 3 (Continued) CTTCGAAACCTACCGGATAGGACTTTACCACGATACCCTTTCCATTTCTATCAAGTCAAT
1 ~ 8~0 L~-uArgAtnL-uAr~lA~pArgS--rLouAllDArDT~rAlDf'h,-A~pPhoTyrCluV~lAsn TCTAAAACTCCTGAAAGACCCCATCAAGCTOTTCCCCACATCAAACCACCACCTCTTACC
3~ 720 SorL~ThrProGluAr~AI-Hi~GluAl~V-lAI-Cln~stLy~Al~Al~Al~LouS~r AATCTTTCTTCAACTGTCTTTCGCCTTAGTCAAATCCTTCCCACCACTAGCCAAGCCACA
A~nV-lS-rS-rS~rVolPh-Gl~LouS-rcluIl~vnlAl~ThrThrs~rGluAl-Thr CTCAACOCCACACTGCACCTCATCTTAATACAAACATCCCACACCTTACTAaCT¢TCAAT
L-uA~n~ lyThrL~uHi-V~ LouIl~GluThrC~-HT-ThrLouL~uClyV-lA~n ACAATGC~CTAAACCCTACCCtC~CTACCT~CGTTATCCCTTCCCTCCCCACCTAATTCT
Thr~o~ClnEnd AATATTTACCACCTTTATTTCATATCTTTACATTTCCACAGTeCGCCTCCCACCTTTAAA

9~1 -------~---------~-------_-~------ _-~_______ _~_________~
CCCTACACTTTATCCTTACTTCTCCAGCACTCCCCTACTCCTCTCCCAACCTTTAGTGTG

AGCCTCTCACCAATAACCTCCACATTACACTCCGTTTCCAAGCCTAAAAAAAAAAAAAAA
1021 ~ 108 AAAA
1081 ---- lOa4 : ~ .

' ~

, -32- ~329~61 ~CHART 4 ' PRV-p C~CTCCAAGAATGAACCTGTGG~TGCTCSTTTCAATGA~AAACTCAAACA ~0 PRV-w CllTCCllAllTG~ TGTlllTACTCCTTTlllTl~ TTIlllGl ~0 CA~GGA~A~TCAG~A~G~AAAGAAA~AGAAA~CAAAAAG~G~A;GAA; 100 11111111 IllllllllllllllllllllI~lllllllilllllllll AAAGCAA~A~C~CAA~CAAAAACA~AAAGAAAAACAA~,iACAGAAAGA~ 0 AAGAccaTGc~AGTGACCGA~ATCATCTCTCA~CTAGCACAA~AACTGG~ 1~0 ~ACACGAT~CTAGTG~CCGAAATGAT~TGTCA~CTACCACAAA~ACTGGA l~O

1111G1a1T1G1G1TaTI11T1TTG1111l11T1G111TTTI11T1TT'C 200 CACAATTAAiTCATTTACTCATAACATCCTTCTACCGAGiATTAACCGCi 250 CACAATTAAATCATTTACTCATAACATCATTCTACCCAG~ATTAACGCAA 250 - AGACTGTCCTT~ATTT~AATCATCTTCTTCAGTACAATCCGC~ACAAATT 300 Il llillllllllllllllll li 11111111 111111111111111 AGTCTGTCCTTA~TTTAA~TC~CCTACTTCACT~TAATCCGCAACAAATT 30~ -.
GAC~TTTCT~ACACTCGTGCCACTCATTCACAATTTG~G~AGTCGTATCA 350 11111111111111111111111111 11111111111111111111111 .
GACATTTCTAACACTCGTGCCACTCACTC~CAATT7CAGAACTCCT~TGA 350 CGG~GTGAGGAATGATTATCGCCTTAATGATAATGAAATGCAAGTC~TGC 400 Illlllllllllllllllilllllll~llllllllllllllllilllllll CCGAGTGAGG~ATCATTATGGCGTTAATaATAATGAA~TGC~AGTGATGC 400 . .
TA~ATCGTTTCATGGTTTGGTCT~TCGACAATCGTACATCTGC~CACATA 460 TlllTGGTTTclTlGTTTccTGTlTcGlcllTGlT~ cTcclGlclTl ~1;0 -, ~
:

.

~33~ ~32~56~
CHART 4 (Continued) TCTCGTG~CTCCGTTATCATCCATGGCCAiACCCAACTTGATTATCCAAT 500 TCTGG7CTCTGGCTTATG~TGGATGGGGA~ACCCAAGT~G~T~A~CCAAT 60~
CAAGCCTTTGATTC~GC~TGCTACTCCGTCATTT~GGCAAATTATGGCT~ 660 II~illlll IllI'lI'lllllllllllllllllillllllllllllllll' CAACCCTTTAATTGAGCATCCTACTCCCTCAtTTAGGCAAATT~T~GCTC 660 ACTTTAGTAiCGCCGCAGAACCATACATTCCGAAGACAAATGCTACTCAG CO0 ACTTTAGTAACGCGGC~G~AGCATACATTGCGA~AAGAAATGCTACTGAG 300 AGCT~CATGCCGCGGTATGGAATC~AGACAAA~TT~C~GACATTAGCCT ~O
Illillllllllllllllllllllllllllllllllllilllllllllll AGGTACATGCCGCGGTATGGAATCA~CAGAAATTTCACTGACATTAGCCT ~50 tGCTAGATACGCTTTCGACTTCtATGAGGTGAATTCGAA~ACACCTGATA 700 tlCTlGlTlCCCTTTCGlCTTCTlTGlCCTGllTTCCllAlClCCTClTl 7 W
GGGCTCGCGAAGCTCACATCCAGATCAAGGCTGCAGCGCTGCCAA~CACC 7~0 1111111111111 11111111111111111111111 11'111111111 .
GCCCTC.CCGAACCCCACATGCAGATGAAGGCTGCA~CACTGCGAAACACT 760 ACTCGCAAAATGTTTCCTATGCACGCCAGTCTTACtAACAACGA~GAAAA 800 ACTCCCACA~TGTTTCGTA~CGACGGCAGTGTTAGTAACAAOGAACAAAA 800 .
CACCGAGAGACACACACTaGAACATCTCAATAGAGACATGCAC:TCTCTCC 850 Illllltlllllllllllllllll ~111111111111111111111111 .
CACGGAGAGACACACACTGCAAGACCTC~ATAGACACATCCACTCTCTCC ~SO
.

~GGCTATGCGCAACTAA C~7 11111~11111111111 TCCCTATCCGCAACT~A ~B7 .

,. . : , ; ': ,' ' ', ' . .' ' '' ' .
' .
., ' ~ ` ~
34 ~ 6 1 S~v SCKEKEGDUDADKDPKKSTSSSKG................ AGTSSKDVNV 34 WUVII SGKETVENLDAGKESKKDASDKGNKPQNSQVGQGSKEPTKTGT~SKDVNV 50 .
S~V GsKGKvv~RLQKITRKMNLp~vEGKIILsLDHLLEyKp~QvDLFNTRATR 84 11111 Illillllllllll 11111111111111111 1111111'1111 W~VII CSKGKEVPRLQKITKK~NLPTVGGKIILSLDHLLEYKPSQVDLFNTRATK 100 SMV TQFEAWYNAVKDEYELDDQ~GVV~NGF~VWCIDNGTSP~NGVWVMMDG 13~
1111 11 111 1111111111111111111111111111 ~11111111 .
WUVII TQFESWYSAVKVEYDLNDEQ~CVIVNCF~VWCIDNGTSPDVNCYWVMMDG 150 S~V rE~IEYPLKPIVENAKPTLRQI~HHFSDAAEAY$EMRNSESPY~PRY~LL lB~
W~VII EEQVEY~LKPIVENAKPTLR~I~HHFSDAAEAYIE~RNSESPY~PRYGLL 200 S~V RNLRDRELARY~FD~YEVTSKTPNRAREAIAQ~KAAALSGVNNKLFGLDG 234 W~VII RNLRDRELARYAFDFYEVTSKTPNRAREATA~KAAALACVN5RLFGLDG 250 , 5~V NISTNSENTERHTARDVN~N~HTLLGVCPiQ 2~6 W4VII NISTNSENTCRHTARDYNQN~H7LLG~CPPC 281 : . .

: , .

~35~ 1329561 pPRV117 * I I *

.

pUC1813/Cpl9 HindIII NcoI TthlllI BstXI NcoI HindIII :
* I I I I tl I ~ 1 1 *
I Pca I Ic I ICMV Coat Protein Genel I Sca pUC1813/Cpl9-PRVexp . HindIII NcoI TthlllI BstXI NcoI HindIII
35 * I ~ *
: I Pca I Ic ¦ CMV I PRV-p Coat I I Sca CoatProtein (16M)6~ne , . , .~ . , . :

.. :.

:
.

-36- ~32~

pGA482 HindIII

10 * ~ ~a~vl ¦ BL ¦
Gus Gen~

pGA482/G/CPl9-PRVexp HindIII TthlllI Bst~I ~indIII

¦Br¦ I ~os I ¦PCalIcl CMV I PRV-p Coat I tscal ¦CaMV¦ IBLI
Coat Protein Gus Protein Ge~e Gene (16AA) ~37~ 132~5~1 CHA~T 8 HindIII NcoI NcoI HindIII
4 ~. ~ 4 . I Pca ¦ Ic I Sca pl8CaMV/CM~-~xp 25. HindIII NcoI HindIII
4 ~ ~ .
* I I I I I --I t *
I Pca ¦ Ic ¦ I Sca , , .

-:, ~
- , . .

-38- ~ 3 2 9 pWMVII-41-3.2 * - L - I - *
10 IWMYII Coat Protein¦
Gene PCR Generated Gene , 20NcoI NcoI

I WMVII Coat Protein Gene pl8WMVII-exp HindIII NcoI NcoI HindIII Ba~HI SmaI
* I I I I , I I I I I I *
35 I PCa I Ic IWMV Coat Protein i I Sca Gene .
.

~39~ ~329~6~

pGA482/G/CPWMVII-exp HindIII NcoI NcoI HindIII

r~ I Nos ¦¦ Pca ¦ Ic ¦ WMVII Coat ¦¦ SCa ¦ ¦ CaMV ¦ IBL¦
Protein G~s Gene Gene . " .

~4~~ ~329~61 pl8GWkNII-exp HindIII NcoI NcoI HindIII BamHI BamHI SmaI

¦ PCa ¦ Ic ¦ WMV Coat l l Sca ~ ¦ CaMV ¦
Protein Gus 15Gene Gene pl8NGWMVII-exp HindIlI NcoI NcoI HindIII BamHI BamHI

IPca ~ WMVII coatl ¦ Sca ~ ¦ CaNV ¦ ¦ Nos ¦-Protein Gus Gene Gene .

~32~56i pZYMV-15 * I I _ * .
ZYMV Coat Proteinl Gene -ZYMV Coat Protein Gene NcoI NcoI
.
IZYMV Coat Protein Gene¦

pUC18CpZYMV

~indIII Ncol NcoI HindlII BamHI SmaI
35 * ~ 1 *
PCa I Ic IZYMY Coat ProteinlI S
Gene :: , .

-42- 1329~61 pGA482/GG/cpZYMV

HindIII NcoI NcoI HindIII

Protein Gus Gene Gene .- ~ .

r ~ ~ :
" . ~ ,, ' ;

- :

-h3- 1 3295 61 pUC18GCpZYMV

HindIII NcoI NcoI HindIII Ba¢HI BamHI SmaI
* Il- I I . 11 1 1 ! I 1... *
¦PCa¦IC¦ZY~V Coat¦¦Scal¦ CaMV j Protein Gu~
Gene Gene pUC18NGCpZYMV

HindIlI NcoI Ncol HlndIII BamHI Ba~H~

¦PCal Ic ¦ZYMV Coat 11 Sca I ¦ CaMV ¦ ¦ Nos ¦
Protein Gus Gene Gene .. . . .

Claims (21)

1. A recombinant DNA molecule comprising a gene which encodes a potyvirus coat protein, said gene selected from the group consisting of:
a) Papaya ringspot virus strain papaya ringspot (PRV-p) coat protein gene having the following nucleotide sequence:

Claim 1 a) Cont'd.

or an equivalent nucleotide sequence encoding for the same amino acids;
b) Watermelon mosaic virus II(WMVII) coat protein gene having the following nucleotide sequence:

Claim 1 b) Cont'd.

Claim 1 b) Cont'd.

or an equivalent nucleotide sequence encoding for the same amino acids; and c) Zucchini yellow mosaic virus (ZYMV) coat protein gene having the following nucleotide sequence:

C1aim 1 c) Cont'd.

or an equivalent nucleotide sequence encoding for the same amino acids.

2. A recombinant DNA molecule according to Claim 1 wherein said recombinant DNA molecule encodes Papaya ringspot virus strain papaya ringspot (PRV-p) coat protein, said recombinant DNA
molecule having the following nucleotide sequence:

Claim 2 Cont'd.

3. A recombinant DNA molecule according to Claim 1 wherein said recombinant DNA molecule encodes Watermelon mosaic virus II
(WMVII) coat protein, said recombinant DNA molecule having the following nucleotide sequence:

Claim 3 Cont'd.

4. A recombinant DNA molecule according to Claim 1 wherein said recombinant DNA molecule encodes Zucchini yellow mosaic virus (ZYMV) coat protein, said recombinant DNA molecule having the following nucleotide sequence:

Claim 4 Cont'd.

5. A recombinant DNA molecule according to Claim 1 further comprising:
a) a promoter;
b) an initiation region; and, c) a poly(A) addition signal;
wherein said promoter is upstream and operably linked to said initiation region, said initiation region is upstream and operably linked to said recombinant DNA molecule encoding a coat protein, and said recombinant DNA molecule encoding a coat protein is upstream and operably linked to said poly(A) addition signal.

6. A recombinant DNA molecule according to Claim 5 wherein said promoter is Cauliflower mosaic virus (CaMV) 35S promoter.

7. A recombinant DNA molecule according to Claim 5 wherein said initiation region is selected from the group consisting of an initiation region derived from the 5' untranslated region of Cucumber mosaic virus (CMV) coat protein gene and an initiation region derived from the 5' untranslated region of SS RUBISCO gene.

8. A recombinant DNA molecule according to Claim 5 wherein said initiation region comprises the sequence AAXXATGG wherein X
is a deoxynucleotide selected from the group consisting of A, C, G and T.

9. A recombinant DNA molecule according to Claim 5 wherein said poly(A) addition signal is selected from the group consisting of: the poly(A) signal derived from Cauliflower mosaic virus (CaMV) 35S gene; the poly(A) signal derived from phaseolin storage protein gene; the poly(A) signal derived from nopaline synthase gene; the poly (A) signal derived from octopine synthase gene; the poly (A) signal derived from bean storage protein gene; and, the poly (A) signal derived from SS RUBISCO.

10. A recombinant DNA molecule according to Claim 6 wherein said initiation region is derived from the 5' untranslated region of Cucumber mosaic virus (CMV) coat protein gene and said poly(A) addition signal is derived from Cauliflower mosaic virus (CaMV) 35S gene.

11. A recombinant DNA molecule according to Claim 5 further comprising an AT rich 5' untranslated region wherein:
a) said AT rich region is downstream from said promoter and upstream from said initiation region;
b) said initiation region comprises the sequence AAXXATGG; and, c) said poly(A) addition signal contains untranslated flanking sequences wherein X is a deoxynucleotide selected from the group consisting of A, C, G and T.

12. A recombinant DNA molecule according to Claim 11 wherein said promoter is Cauliflower mosaic virus (CaMV) 35S promoter.

13. A recombinant DNA molecule according to Claim 11 wherein said AT-rich 5' untranslated region is derived from the 5' untranslated region of a gene selected from the group consisting of Cucumber mosaic virus (CMV) coat protein gene and SS RUBISCO
gene.

14. A recombinant DNA molecule according to Claim 11 wherein said initiation region is derived from the 5' untranslated region of a gene selected from the group consisting of Cucumber mosaic virus (CMV) coat protein gene and SS RUBISCO gene.

15. A recombinant DNA molecule according to Claim 11 wherein said poly(A) addition signal is selected from the group consisting of: the poly(A) signal derived from Cauliflower mosaic virus (CaMV) 35S gene; the poly(A) signal derived from phaseolin storage protein gene; the poly(A) signal derived from nopalinesynthase gene; the poly(A) signal derived from octopine synthase gene; the poly(A) signal derived from bean storage protein gene; and, the poly(A) signal derived from SS RUBISCO.

16. A recombinant DNA molecule according to Claim 12 wherein:
a) said AT rich 5' untranslated region and said initiation region are derived from the 5' untranslated region of Cucumber mosaic virus (CMV) coat protein gene;
b) said initiation region comprises the sequence AAXXATGG; and, c) and said poly(A) addition signal is derived from Cauliflower mosaic virus (CaMV) 35S gene wherein X is a deoxynucleotide selected from the group consisting of A, C, G
and T.

17. A transformed plant cell containing a recombinant DNA
molecule according to Claim 5.

18. A transformed plant cell comprising a recombinant DNA
molecule according to Claim 10.

19. A transformed plant cell comprising a recombinant DNA
molecule according to Claim 11.

20. A transformed plant cell comprising a recombinant DNA
molecule according to Claim 16.

21. A process for producing a transgenic plant which is resistant to viral infection comprising the steps of:
a) constructing a recombinant DNA molecule according to Claim 5;
b) transforming plant cells with said recombinant DNA;
and c) regenerating plants from said transformed plant cells.